a-Fetoprotein (AFP) and Albumin (AIb) are closely related serum proteins, encoded by a coordinately regulated chromosomal locus. AFP is developmentally regulated, expressed in fetal liver, silenced at birth, but reactivated in liver cancer. AIb characterizes hepatocyte specification and its expression persists throughout life. Investigators have long understood that the mechanisms that regulate the AIb-AFP locus will define the normal, fetal, and neoplastic hepatocyte phenotype. Both genes are primarily regulated by distant enhancers, while AFP silencing is mediated by promoter controls. Previous experiments have demonstrated that each promoter interacts with enhancers through a different mechanism. In the AIb _romoter, HNF1 is the critical enhancer-activating factor. Aim 1 experiments will define the specific HNF1 "lomains that mediate this interaction and the cofactors that these domains recruit. Further studies will characterize undefined elements in the rest of the promoter, especially "architectural" factors that facilitate enhancer activation. Aim 2 will focus on the AFP promoter, facilitated by use of a synthetic promoter designed for easy modification. The silencer will be localized; its binding factors and mechanism will be characterized. The potential roles of 6 new transcription factors will also be assessed. These were identified by their coregulation with AFP in microarray studies. Aim 3 will integrate the entire locus into a novel cell transfection system to study enhancers and promoters in their proper chromosomal relationships. The entire 70-kb locus has been slightly modified to clone it into a set of easily manipulated plasmids that can be joined together or experimentally changed. These have been placed into a recombining plasmid that will insert constructs into human HCC cell lines. Cre-mediated site-specific recombination will incorporate a single gene copy into a specific chromosomal location. The system will be used to define the relationships of individual enhancers and promoters, a set of new regulatory elements identified in sequence analysis, and specific transcriptional mechanisms.